Deliberate acts of terrorism against the United States demonstrated the importance of equipping our nation with the means to defend against these actions. The anthrax letters in 2001 had severe impacts on human health, resulting in 11 hospitalizations, 5 deaths, and fear based on inability to rapidly diagnose. In response to these attacks, Idaho Technology Inc (ITI) created diagnostic systems capable of detecting biowarfare agents. However, the 2007 NIAID Strategic Plan for Biodefense indicates the need to migrate from the "one bug, one solution" approach, to methods capable of responding to multiple threat organisms. For example, there is a need to identify more than a dozen biowarfare agents and distinguish them from common pathogens that produce similar life threatening critical illness. Another need is for highly adaptable methods, which allow rapid modification as new biothreat agents emerge. ITI is meeting these needs by developing a new system called the FilmArray(tm), which comprises a real-time polymerase chain reaction (PCR) instrument and novel lab-in-a-pouch that performs all steps from sample purification to multiplex organism detection and identification in under 1 hour. A single human sample is injected into the pouch, then inserted in the instrument which automatically performs all required steps. This system is highly adaptable, as pouches can be manufactured with different multiplex assays. Thus far we have developed a pouch that can identify 20 respiratory pathogens, in a 120-well array. In this proposal, ITI will extend FilmArray's capabilities by developing a system that identifies 32 organisms. To do so the following aims will be pursued. Aim 1. Produce FilmArray biothreat assays that identify both biowarfare agents and common pathogens. Aim 2. Increase the number of organisms detected by building a 224-well 2nd stage PCR array. Aim 3. Develop methods for FilmArray automated isolation of bacterial and viral DMA and RNA from nasal swabs and cerebral spinal fluid (CSF). Aim 4. Develop software to facilitate the automated identification of pathogen(s) within a specimen. Aim 5. Validate the FilmArray biothreat system with human samples, including nasal swabs, blood, and CSF at ITI, external laboratories and hospitals. RELEVANCE (See instructions^: The FilmArray will fill a need for rapid, sensitive, specific, easy to use, adaptable, and cost-effective medical diagnostics for public health and hospital-based clinical laboratories, and point-of-care use. FilmArray's automated and simultaneous detection of multiple pathogens will distinguish individuals infected by a biothreat agent or a common infection with similar, generalized symptoms.